Key Benefits

Why is it Important to Verify Hematopoietic Progenitor Cell Identity using Established Markers?

Colony forming cell (CFC) assays, which are used to enumerate and quantify multi-potent and single lineage hematopoietic progenitors, can be time consuming and laborious.

Successful growth and enumeration of cell colonies is dependent on factors such as accurate cell counts, the presence of growth factors and/or cytokines, adequate humidity, and the use of high quality media. R&D Systems offers Human Methylcellulose Base Media with superior optical clarity to support optimal colony growth, enumeration, and identification. The Human Methylcellulose Base Media contains components that have been optimized for CFC assays. Individual researchers can customize the media by adding cells and other culture supplements tailored to their specific research. This product can also be used in the long-term culture-initiating cell (LTC-IC) assay.

Human Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay. A. Colony forming unit-erythroid (CFU-E) are clonogenic progenitors that produce only one or two clusters with each cluster containing from 8 to approximately 100 hemoglobinized erythroblasts. It represents the more mature erythroid progenitors that have less proliferative capacity. B. Colony forming unit-granulocyte (CFU-G) are clonogenic progenitors of granulocytes that give rise to a homogeneous population of eosinophils, basophils, or neutrophils. C. Colony forming unit-granulocyte, macrophage (CFU-GM) are progenitors that give rise to colonies containing a heterogeneous population of macrophages and granulocytes. The morphology is similar to the CFU-M and CFU-G descriptions. D. Burst forming unit-erythroid (BFU-E) colonies can be described as small (3 to 8 clusters), intermediate (9 to 16 clusters), or large (more than 16 clusters) according to the number of clusters present. These are primitive erythroid progenitors that have high proliferative capacity. E. Colony forming unit-macrophage (CFU-M) are clonogenic progenitors of macrophages that give rise to a homogenous population of macrophages. F. Colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) are multi-lineage progenitors that give rise to erythroid, granulocyte, macrophage and megakaryocyte lineages, as the name indicates.

Background: Hematopoietic Stem Cells

The definitive hematopoietic system is made up of all adult blood cell types including megakaryocytes, erythrocytes, and cells of the myeloid and lymphoid lineages. All of these cells are derived from multipotent hematopoietic stem cells (HSCs) through a succession of precursors with progressively limited potential. Hematopoietic stem cells are tissue-specific stem cells that exhibit remarkable self-renewal capacity and are responsible for the life-long mainte­nance of the hematopoietic system. HSCs are rare cells that reside in adult bone marrow where hematopoiesis is continu­ously taking place. They can also be found in cord blood, fetal liver, adult spleen, and peripheral blood. R&D Systems offers several products for studying hematopoietic lineage cells including serum-free media, lineage deple­tion antibodies and kits, and reagents for performing colony forming cell (CFC) assays.

Transfer the appropriate volume of cells plus a slight excess into a new 15 mL centrifuge tube.

Centrifuge at 300 x g for 5 minutes.

Remove the supernatant.

Resuspend the cells in Cell Resuspension Solution to the desired stock cell number to generate a 10X stock concentration.

Combine the appropriate volume of 10X cell stock with the desired cell culture supplements/cytokines, and Human Methylcellulose Base Media. The final methylcellulose concentration should be 1.27%.

Vortex the samples vigorously.

Wait approximately 20 minutes to allow air bubbles to escape.

Add 1.1 mL of the cell mixture to a 35 mm culture plate using a 3 mL syringe and a 16 gauge needle.

Spread the media evenly by gently rotating the plate.

Place two 35 mm plates into a 10 cm plate.

Add one uncovered 35 mm plate that contains 3-4 mL of sterile water.

Cover the 10 cm plate and place it in a 37 °C and 5% CO2 incubator.

Incubate the cells for 14-16 days.

Use an inverted microscope and a scoring grid to identify and count individual colonies.

Citations for Human Methylcellulose Base Media

R&D Systems personnel manually curate a database that contains references using R&D Systems products.
The data collected includes not only links to publications in PubMed,
but also provides information about sample types, species, and experimental conditions.

FAQs

What is the difference between Methylcellulose Stock (Catalog # HSC001) and Base Media (Catalog # HSC002)?

The Methylcellulose Stock (Catalog # HSC001) contains methylcellulose in Iscove's Modified Dulbecco's Medium. The Base Media (Catalog # HSC002) contains methylcellulose in Iscove's Modified Dulbecco's Medium along with serum, albumin, L-glutamine, and 2-mercaptoethanol. The additional components of HSC002 are needed to sustain the viability and growth of HSCs. The advantage to using HSC002 is that R&D Systems has already evaluated the serum, albumin, L-glutamine, and 2-mercaptoethanol in-house to ensure lot-to-lot consistency, eliminating the need for the researcher to evaluate these components individually.

Can the CFU assay using Methycellulose based media be performed using frozen PBMCs instead of fresh PBMCs?

Yes, the CFU assay can be performed using frozen PBMCs. The PBMCs can be frozen in DMEM containing 10% FBS and 10% DMSO.